Study On The Determination Oiorganophosphorus And Carbamate Pesticides Based On Enzyme Biosensors

Pesticide residue that threatens people’s living environment and heath seriously is asevere problem the contemporary society faces. Thus, carrying out fast and sensitive analysisto pesticide residue has great significance for protection of ecological environment andpeople’s life security. Among many analysis methods, electrochemical biosensor drawspeople’s wide concern due to its simple equipment, low cost, fast response and highsensitivity. In this study, three electrochemical enzymatic biosensors were constructed basedon new nanomaterials and their application in organophosphorus and carbamate pesticidesdetermination was investigated. The main contents could be outlined as follows:（1） A novel tyrosinase （Tyr） biosensor was prepared by immobilizing tyrosinase ongraphene-nano Pt composites modified electrode via electrostatic interaction withself-assembled monolayer of cysteamine. Scanning electron microscope （SEM）, cyclicvoltammetry （CV） and electrochemical impedance spectroscopy （EIS） were employed tocharacterize the materials and the modified electrodes. The results showed that graphene-nanoPt composites greatly enhanced the electron transfer with the sensitivity of334mA M-1tocatechol. The immobilized Tyr remained relatively high bioactivity and exhibited high affinityto catechol with the K^app_mvalue of27.6M. Under optimum conditions, chlorpyrifos,profenofos and malathion were detected using chronoamperometric method based on theirinhibition effect on Tyr activity. The liner ranges were0.25-10ppb,1-10ppb and5-30ppbwith the detection limits of0.2,0.8and3ppb for chlorpyrifos, profenofos and malathion,respectively.（2） A novel acetylcholinesterase （AChE） biosensor was developed based on immobilizingAChE on3-carboxyphenylboronic acid （CPBA）/graphene-gold nanocomposites modifiedelectrode via the specific binding between the boronic acid group of CPBA and the glycosylof AChE. Transmission electron microscopy （TEM）, SEM, CV and EIS were used tocharacterize the materials and the modified electrodes. The biosensor enjoyed good sensitivityowing to the excellent properties of gold nanoparticles （AuNPs） and graphene （GR） whichpromoted electron transfer reaction and enhanced the current response. The immobilizedAChE remained relatively high activity and exhibited high affinity to substrate with the K^app_mvalue of0.16mM. Under the optimal conditions, chlorpyrifos, malathion, carbofuran andisoprocarb were determined using chronoamperometric method based on their inhibitioneffect on AChE activity. The detection limits were0.1ppb,0.5ppb,0.05ppb and0.5ppb forchlorpyrifos, malathion, carbofuran and isoprocarb respectively. （3） A new AChE biosensor was developed by immobilizing AChE on boronic acidfunctionalized Au@Fe magnetic nanoparticles/graphene-alginate nanocomposites modifiedelectrode. Graphene-alginate composites modified electrode was firstly prepared as the matrix.Then boronic acid functionalized Au@Fe magnetic nanoparticles were anchored by thecovalence between the cis-diol of alginate and the boronic acid group on Au@Fenanoparticles. AChE was subsequently immobilized via the bonding between the glycosyl ofacetylcholinesterase and the boronic acid group. TEM, EIS and CV were used to characterizethe materials and the modified electrodes. The excellent properties of alginate-graphenecomposites and boronic acid functionalized Au@Fe magnetic nanoparticles guaranteed thehigh sensitivity of the biosensor. Besides, their good biocompatibility also provided afavorable microenvironment for enzyme immobilization. The immobilized AChE retainedrelatively high bioactivity and exhibited high affinity to substrate with the K^app_mvalue of44M. Based on enzyme inhibition, carbofuran was detected using square wave voltammetry（SWV）. Good relationships were obtained between the inhibition percentage and pesticideconcentration in the ranges of0.05-15ppb and15-400ppb with a detection limit of0.01ppb.